1) Alzheimers disease (AD) involves the abnormal accumulation of tau filaments in the brain. To probe the molecular basis for faithful AD tau filament propagation and to improve detection of tau aggregates as potential etiological biomarkers, we exploited the seeded polymerization growth mechanism of tau filaments to develop a highly selective and ultrasensitive cell-free AD tau seed amplification assay (AD RT-QuIC). AD RT-QuIC is performed in multi-well plates with fluorescence detection. The reaction is based on the ability of AD tau aggregates to seed the formation of amyloid fibrils with certain recombinant tau fragments, including one that spans the known amyloid core of the straight and paired helical tau filaments of AD. AD RT-QuIC detected seeding activity in AD brains at dilutions as extreme as 10E7-10E10-fold but was 10E2-10E6-fold less responsive when seeded with brain from most cases of other types of neurodegenerative disease, including other types of tauopathies with comparable loads of aggregated tau in the brain. For example, AD brains had average AD RT-QuIC seeding activities that were orders of magnitude higher than Picks disease brains, but the opposite was true using our previously described Pick-optimized tau RT-QuIC assay. Consistent with the AD seeds being tau filaments, we found that, like the tau filaments of AD brain, they were Sarkosyl-insoluble, protease-resistant, and reactive with tau antibodies. Moreover, AD RT-QuIC detected as little as 7 fg of pure synthetic tau fibrils. The profoundly distinctive seeding activity exhibited by AD tau filaments in these seeded polymerization reactions provides a mechanistic basis for their consistent propagation as specific conformers in AD patients. Importantly, the AD RT-QuIC assay also provides rapid ultrasensitive quantitation of AD tau seeding activity as a biomarker. 2) Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common prion disease in humans and has been iatrogenically transmitted through corneal graft transplantation. Approximately 40% of sCJD patients develop visual or oculomotor symptoms and may seek ophthalmological consultation. We used the highly sensitive real time quaking-induced conversion (RT-QuIC) assay to measure post-mortem prion levels in cornea, lens, ocular fluid, retina, choroid, sclera, optic nerve, and extraocular muscle in the largest series of sCJD patient eyes studied by any assay to date. We detected prion seeding activity in 100% of sCJD eyes representing three common sCJD subtypes with levels varying by up to 4 log-fold among individuals. The retina consistently showed the highest prion levels, which in some cases were only slightly lower than brain. Within the retina, prion deposits were detected by immunohistochemistry in the outer plexiform layer, and in some eyes the inner plexiform layer, consistent with synaptic prion deposition. Prion seed levels by RT-QuIC generally declined in eye tissues with increased distance from the brain, and all corneas had prion seeds detectable. Prions were also present in the lens and sclera. Collectively, these results reveal that sCJD patients accumulate prions throughout the eyes, indicating the potential diagnostic utility as well as the possible biohazard. 3) A definitive pre-mortem diagnosis of prion disease depends on brain biopsy for prion detection currently and no validated alternative preclinical diagnostic tests have been reported to date. To address the feasibility of using skin for preclinical diagnosis, we report ultrasensitive serial protein misfolding cyclic amplification (sPMCA) and real-time quaking-induced conversion (RT-QuIC) assays of skin samples from hamsters and humanized transgenic mice (Tg40h) at different time points after intracerebral inoculation with 263K and sCJDMM1 prions, respectively. sPMCA detected skin PrPSc as early as 2 weeks post inoculation (wpi) in hamsters and 4 wpi in Tg40h mice; RT-QuIC assay revealed earliest skin prion-seeding activity at 3 wpi in hamsters and 20 wpi in Tg40h mice. Unlike 263K-inoculated animals, mock-inoculated animals showed detectable skin/brain PrPSc only after long cohabitation periods with scrapie-infected animals. Our study provides the proof-of-concept evidence that skin prions could be a biomarker for preclinical diagnosis of prion disease.